2-Deoxyfortimicin B

ABSTRACT

A new fortimicin antibiotic, 2-deoxyfortimicin B, intermediates useful in the preparation of the compound, methods of making and using the compound and compositions containing 2-deoxyfortimicin B.

BACKGROUND OF THE INVENTION

Despite the availability of a variety of highly effective antibiotics,the search for new antibiotics is a continuing one. The primary reasonfor the continuing search is the reoccurring development ofmicroorganisms which are resistant to existing antibiotic therapy. Thus,there is a continuing need for new antibiotics which are eitherintrinsically more active than existing drug entities and thus can beadministered in lower dosages to minimize the side effects of thesepowerful drugs, or are effective against resistant strains.

A number of aminoglycoside antibiotics are known, such as the gentamicinand kanamycin family of antibiotics. More recently, a new family ofaminoglycosides, the fortimicins have been identified. See, for example,U.S. Pat. Nos. 3,976,768 and 3,931,400.

Although the fortimicin family is a relatively new group of antibiotics,clinical experience has shown that aminoglycoside antibiotics aresusceptible to resistant strain development. In many cases, theresistance is R-factor mediated and is attributed to the ability of thebacteria to enzymatically modify the amino or hydroxy groups of theaminoglycoside antibiotics.

Thus, there is a continuing need for new antibiotic entities in thisvaluable antibiotic family. The present invention provides one suchentity.

SUMMARY OF THE INVENTION

This invention relates to a new, synthetic fortimicin derivative,2-deoxyfortimicin B. In addition to its antibiotic properties, thecompound of this invention is useful as an intermediate in the synthesisof 2-deoxyfortimicin A. Intermediates are also provided as well aspharmaceutical compositions and methods.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The compound of this invention, 2-deoxyfortimicin B, is represented byFormula I: ##STR1##

2-Deoxyfortimicin B exhibits anti-bacterial activity against variousGram-positive and Gram-negative bacteria. The antibiotic can be employedin daily dosages of from 10 to 200 mg/kg, by parenteral routes ofadministration, i.e., by intramuscular, intravenous, subcutaneous andintraperitonal routes, for a period of time sufficient to provide anantibiotic effect, generally, for a period of time following thedisappearance of infection symptoms. Additionally, the anti-bacterialagent of this invention can be incorporated into anti-bacterial ordisinfecting solutions which are used as a surface disinfectant forcontrolling the population of various bacteria.

2-Deoxyfortimicin B is also useful as an intermediate for2-deoxyfortimicin A which is a new antibiotic disclosed in our copendingU.S. patent application concurrently filed and coassigned.

The pharmaceutically acceptable salts of 2-deoxyfortimicin B are alsoincluded within the scope of this invention. The term "pharmaceuticallyacceptable salts" refers to non-toxic acid addition salts which areprepared by reacting the base with a suitable organic or inorganic acid.Representative salts include the hydrochloride, hydrobromide, sulfate,bisulfate, acetate, oxalate, valerate, oleate, palmitate, stearate,laurate, borate, benzoate, lactate, phosphate, tosylate, citrate,maleate, fumarate, succinate, tartrate, napsylate and the like salts.

The present invention further provides new derivatives of fortimicin Buseful as intermediates in preparing 2-deoxyfortimicin B and representedby Formulae II, III and IV.

Compounds of formula II are 2-O-hydrocarbonsulfonyl derivativesrepresented as follows: ##STR2## wherein X is a straight, branched orcyclic saturated or unsaturated hydrocarbon moiety having from 1 to 8carbon atoms and Z is benzyloxycarbonyl;

Compounds of Formula III are the cyclic 4,5-oxazolidine derivatives ofthe compounds of formula II: ##STR3## wherein X is as defined above, Zis benzyloxycarbonyl and R is hydrogen, loweralkyl, aryl or substitutedaryl.

The 2-O-hydrocarbon sulfonyl derivatives are those derived fromhydrocarbon sulfonic acid having up to 8 carbon atoms, such asmethanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid,toluenesulfonic acid, i.e., compounds of Formulae II and III wherein Xis lower alkyl, loweralkenyl, lower alkynyl, aryl, aralkyl and the like.

The term "lower alkyl" refers to straight or branched chain alkylradicals having from 1 to 6 carbon atoms, i.e., methyl, ethyl, n-propyl,iso-propyl, n-butyl, sec-butyl, tert-butyl, n-pentyl, 2-methylbutyl,n-hexyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl and the like.

The term "loweralkenyl" refers to C₂ to C₆ alkenyl moieties, i.e.,ethenyl, propenyl, n-butenyl, n-pentenyl, n-hexenyl and the like.

The term "loweralkynyl" refers to C₂ to C₆ alkynyl groups, i.e.,ethynyl, propynyl, 2-butynyl, 2-pentynyl, etc.

"Aryl" as used herein, refers to aromatic hydrocarbons such as benzyl,phenyl, tosyl, toluyl and the like.

Aralkyl includes, but is not limited to aromatic hydrocarbons havinglower alkyl sidechains, i.e., 1-methylphenyl, 2-butylphenyl, and thelike.

1,2-epiminofortimicin B is also provided by the present invention. Thecompound is represented by Formula IV: ##STR4##

2-deoxyfortimicin B can be prepared as follows. In one process,fortimicin B, having all primary amino groups protected bybenzyloxycarbonyl groups and the C₅ hydroxyl and C₄ secondary aminogroup blocked by a suitable aldehyde to form an oxazolidine ring, upontreatment with a hydrocarbonsulfonyl halide or anhydride, is convertedto a 2-O-methanesulfonyl ester (e.g., to a 2-O-methanesulfonyl ester)which in turn is converted to a1,2',6'-tri-N-benzyloxycarbonyl-2-O-hydrocarbonsulfonyl ester derivativefollowing acid hydrolysis of the oxazolidine ring, which is N-deblockedby catalytic hydrogenolysis in the presence of an acid. When theresulting 2-O-hydrocarbonsulfonylfortimicin B salt is converted to thefree base, the intermediate 1,2-epiminofortimicin B is obtained.Continuing the process, catalytic hydrogenolysis of1,2-epiminofortimicin B gives 2-deoxyfortimicin B which in turn isconverted is 2-deoxyfortimicin A.

In an alternate procedure the key intermediate 1,2-epiminofortimicin Bis conveniently prepared as follows. Fortimicin B, having all primaryamino groups protected by Schiff base formation from a suitable aldehyde(e.g., benzaldehyde) and the C₅ hydroxyl and C₄ secondary amino groupprotected by the same aldehyde to form an oxazolidine ring, upontreatment with a hydrocarbonsulfonyl halide or anhydride, is convertedto a 2-O-hydrocarbonsulfonyl ester which in turn is converted, on acidhydrolysis of the Schiff base and oxazolidene to a2-O-hydrocarbonsulfonylfortimicin B salt. The salt upon conversion tothe free base rearranges to 1,2-epiminofortimicin B.

The following Examples further illustrate the present invention.

EXAMPLE 1 1,2',6'-Tri-N-benzyloxycarbonylfortimicin B

To a stirred solution of 2.0 g of fortimicin B 30 ml of water and 60 mlof methanol, cooled in an ice bath, is added 4.44 g ofN-(benzyloxycarbonyloxy)succinimide. Stirring is continued at 0° for 3hours and then at room temperature for 22 hours. The major portion ofthe methanol is evaporated under reduced pressure and the residue isshaken with a mixture of chloroform and water. The chloroform solutionis washed with water and dried over anhydrous magnesium sulfate. Thechloroform is evaporated and the residue is chromatographed on silicagel. Elution with a solvent system composed ofchloroform-methanol-ammonium hydroxide (234:1.4:0.1 v/v/v) gives 1.05 gof 1,2',6'-tri-N-benzyloxycarbonylfortimicin B: [α]_(D) ²⁵ -16.5° (c1.0, CH₃ OH); IR (CDCl₃) 1712 and 1507 cm⁻¹ ; NMR (CDCl₃) 1.03 (C_(6')--CH₃, J_(6'),7' =6.0 Hz), 2.32 (C₄ --NCH₃), 3.41 (OCH₃).

Anal. Calcd. for C₃₉ H₅₀ N₄ O₁₁ : C, 62.39; H, 6.71; N, 7.46. Found: C,62.16; H, 6.76; N, 7.43.

EXAMPLE 2 1,2',6'-Tri-N-benzyloxycarbonyl-4,5-salicylaldehydeoxazolidine fortimicin B

A solution of 22 g of 1,2'6'-tri-N-benzyloxycarbonylfortimicin B in 396ml of methanol is treated with 3.96 ml of salicylaldehyde and refluxedfor 1 hour. Evaporation of the reaction mixture under reduced pressuregives 26 g of 1,2',6'-tri-N-benzyloxycarbonyl-4,5-salicylaldehydeoxazolidine fortimicin B as a brownish yellow solid: NMR (CDCl₃) δ 0.94(C₆ '--CH₃, J_(6'),7' =7.0 Hz), 2.34 (C₄ --NCH₃), 3.49 (C₃ --OCH₃), 7.31(Cbz)

EXAMPLE 31,2',6'-Tri-N-benzyloxycarbonyl-4,5-(2-O-methanesulfonylsalicylaldehyde)-oxazolidine-2-O-methanesulfonylfortimicinB

A stirring solution of 26 g of1,2',6'-tri-N-benzyloxycarbonyl-4,5-salicylaldehyde oxazolidinefortimicin B in 154 ml of dry pyridine is treated with 12.26 ml offreshly distilled methanesulfonylchloride. After stirring for 20 hoursthe reaction mixture is poured into 2000 ml of 5% sodium hydrogencarbonate solution and extracted 2 times with 1000 ml portions ofchloroform. The combined chloroform extract is washed with 1000 ml of 5%sodium hydrogen carbonate and then twice with 1000 ml portions of water.The chloroform is evaporated under reduced pressure and the pyridine isremoved by repeated co-distillation with benzene to give 31.2 g of1,2',6'-triN-benzyloxycarbonyl)-4,5-(2,O-methanesulfonylsalicylaldehyde)-oxazolidine-2-O-methanesulfonylfortimicinB: NMR (CDCl₃) δ 1.0 (C_(6') --CH₃, J_(6'7') =7.0 Hz), 2.19 (C₄ --NCH₃),2.94 (C₂ --OSO₂ CH₃), 3.15 (Ar--OSO₂ CH₃), 3.60 (C₃ --OCH₃), 7.33 (Cbz).

EXAMPLE 4 1,2',6'-Tri-N-benzyloxycarbonyl-2-O-methanesulfonylfortimicinB

A stirring solution of 31.2 g of1,2'6'-tri-N-benzyloxycarbonyl-4,5-(2-O-methanesulfonylsalicylaldehyde)oxazolidine-2-O-methanesulfonylfortimicin B in 1000 ml oftetrahydrofuran is treated with 262 ml of 0.4 N hydrochloric acid. Afterstirring for 4 hours, the reaction mixture is poured into 5700 ml of 6 Nammonium hydroxide solution and extracted 2 times with 1400 ml portionsof chloroform. The combined chloroform extract is washed with 5700 ml of7% sodium hydrogen sulfite solution and then 2 times with 1180 mlportions of water. Removal of the chloroform under reduced pressuregives 27.35 g. of crude1,2',6'-tri-N-benzyloxycarbonyl-2-O-methanesulfonylfortimicin B. Thecrude material is chromatographed on a column (6.0×80 cm) of SephadexLH-20 gel prepared and eluted with 95% ethanol. Fractions containing thedesired material are combined and concentrated to dryness under reducedpressure to give pure1,2',6'-tri-N-benzyloxycarbonyl-2-O-methanesulfonylfortimicin B as aglass: [α]_(D) ²³ + 18.5° (c 1.0, CH₃ OH); IR (CDCl₃) 3436, 3350, 1703,1502, 1354 and 1173 cm⁻¹ ; NMR (CDCl₃) δ 1.07 (C₆ --CH₃, J_(6'),7' =7.0Hz) 2.34 (C₄ --NCH₃), 2.87 (OSO₂ CH₃), 3.48 (OCH₃).

Anal. Calcd. for C₄₀ H₅₂ N₄ O₁₃ S: C, 57.96; H, 6.32; N, 6.76. Found: C,57.65; H, 6.52; N, 6.62.

EXAMPLE 5 2-O-Methanesulfonylfortimicin B Tetrahydrochloride

A solution of 4.42 g of1,2',6'-tri-N-benzyloxycarbonyl-2-O-methanesulfonylfortimicin B in 310ml of 0.2 N hydrochloric acid in methanol is treated for 4 hours with4.5 g of 5% palladium on carbon under hydrogen and 3 atmospheres ofpressure. The catalyst is filtered off and washed with ethanol. Thefiltrate is concentrated to dryness under reduced pressure and theexcess hydrochloric acid is removed by repeated co-distillation withmethanol to leave 2.79 g of 2-O-methanesulfonylfortimicin Btetrahydrochloride as a white glass: [α]_(D) ²⁵ +91.7° (c 1.01, CH₃ OH);IR (KBr) 3400, 2920, 1590, 1330 and 1165 cm⁻¹ ; NMR (D₂ O) δ 1.82(C_(6') --CH₃, J_(6'7') =7.0 Hz), 3.31 (C₄ --NCH₃), 3.88 (C₂ --OS₂ CH₃),4.07 (C₃ --OCH₃), 5.88 (C_(1') H, J=4.0 Hz)

EXAMPLE 6 1,2-Epiminofortimicin B

A solution prepared from 2.8 g of 2-O-methanesulfonylfortimicin Btetrahydrochloride in 20 ml of water is passed through a column (2.2×20cm) of an anion exchange resin quarternary ammonium styrene type, e.g.,AG®2-X8, 50-100 mesh resin, (OH form) sold by Bio-Rad laboratories,sufficient to remove the chloride ion. Basic elutes are combined andallowed to stand at room temperature for 72 hours. Evaporation of thewater under reduced pressure leaves 3.0 g of 1,2-epiminofortimicin B:NMR (D₂ O) δ 1.55 (C_(6') --CH₃, J_(6'),7' =7.0 Hz), 2.83 (C₄ --NCH₃),4.02 (C₃ --OCH₃), 5.42 (C_(1') H, J=3.0 Hz).

EXAMPLE 7 2-Deoxyfortimicin B and1-Deamino-2-deoxy-2-epi-aminofortimicin B

A solution prepared from 3.22 g of 1,2-epiminofortimicin B in 250 ml ofwet ethanol is treated for 24 hours with 12 g of Raney nickel under 3atmospheres of hydrogen. The catalyst is collected on a filter andwashed with ethanol. The filtrate is concentrated to dryness underreduced pressure to give 2.90 g of a mixture of 2-deoxyfortimicin B and1-deamino-2-deoxy-2-epi-aminofortimicin B as a white froth. The mixtureis chromatographed on a column (2.9×50 cm) of a cation exchange resin,(NH₄ ⁺ form) (e.g., BioRad 70 100-200 mesh, carboxylic styrene typeresin sold by Bio-Rad Laboratories, and eluted with a gradient of waterto 1 N ammonium hydroxide. The first elutes are taken to dryness underreduced pressure to yield 1.347 g of pure 2-deoxyfortimicin B: NMR (D₂O) δ 1.5 (C_(6') --CH₃, J_(6'),7' =7.0 Hz), 2.82 (C₄ --NCH₃), 3.86 (C₃--OCH₃), 5.48 (C₁, H, J=3.5 Hz).

Later elutes are collected and taken to dryness under reduced pressureto yield 1.172 g of 1-deamino-2-deoxy-2-epi-aminofortimicin B: NMR (D₂O) δ 1.51 (C_(6') CH_(3') J_(6'),7' =7.0 Hz), 2.83 (C₄ --NCH₃), 4.02 (C₃--OCH₃), 5.31 (C_(1') H, J=4.0 Hz).

The in vitro antibiotic activity of 2-deoxyfortimicin B is determined bya two-fold dilution test using Mueller-Hinton agar, 10 ml per Petriplate. The inoculum of approximately 1×10⁵ of the indicated testorganism is delivered by the Steer's replicator. The test is incubatedat 37° C. for 24 hours.

The compounds of this invention are active as systemic antibiotics wheninjected by parenteral routes of administration, i.e., by theintramuscular, intravenous, intraparitoneal or subcutaneous routes ofadministration. The compounds can also be administered orally in thoseinstances where it is desirable to sterilize the intestinal tract andcan additionally be applied topically or rectally.

Solid dosage forms for oral administration include capsules, tablets,pills, powders and granules. In such solid dosage forms, the activecompound is admixed with at least one inert diluent such as sucrose,lactose or starch. Such dosage forms can also comprise, as is normalpractice, additional substances other than inert diluents, e.g.,lubricating agents such as magnesium stearate. In the case of capsules,tablets and pills, the dosage forms may also comprise buffering agents.Tablets and pills can additionally be prepared with enteric coatings.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups and elixirscontaining inert diluents commonly used in the art, such as water.Besides, such inert diluents, compositions can also include adjuvants,such as wetting agents, emulsifying and suspending agents, andsweetening, flavoring and perfuming agents.

Preparations according to this invention for parenteral administrationinclude sterile aqueous or non-aqueous solutions, suspensions, oremulsions. Examples of non-aqueous solvents or vehicles are propyleneglycol, polyethylene glycol, vegetable oils, such as olive oil, andinjectable organic esters such as ethyl oleate. Such dosage forms mayalso contain adjuvants such as preserving, wetting, emulsifying, anddispersing agents. They may be sterilized, by for example, filtrationthrough a bacteria-retaining filter, by incorporating sterilizing agentsinto the compositions. They can also be manufactured in the form ofsterile solid compositions which can be dissolved in sterile water, orsome other sterile injectable medium immediately before use.

Compositions for rectal administration are preferably suppositorieswhich may contain, in addition to the active substance, excipients suchas cocoa butter or a suppository wax.

The dosage of active ingredient in the compositions of this inventionmay be varied; however, it is necessary that the amount of the activeingredient shall be such that a suitable dosage form is obtained. Theselected dosage depends upon the desired therapeutic effect, on theroute of administration, and on the duration of the treatment.Generally, dosage levels of between 10 to 200 mg/kg of body weight dailyare administered to a mammalian patient suffering from an infectioncaused by susceptible organism.

We claim:
 1. 2-Deoxyfortimicin B or a pharmaceutically acceptable salt thereof. 